The present invention relates to a method for removing solvent or game generally liquid from a liquid sample containing macromolecules or for washing said molecules i.e. replacing the solvent and a device for carrying out this method.
There has been a number of analytical procedures developed in the biochemical art wherein it is required to remove solvent from e.g. protein solutions in order to have a more concentrated protein sample which can be analyzed effectively, or to remove low molecular weight ions or solutes. Many other analytical procedures involving not only proteins but macromolecular species in general, have also been developed wherein it is necessary to concentrate a macromolecular component in a liquid sample.
When concentrating small volumes of macromolecules in solution using an absorbent media which draws solute through a filter in the form of a thin membrane by capillary action there exists the problem of achieving a sufficiently fast filtration which is in contrast with the need to reduce the effective membrane area in order to minimize loss of macromolecular material which tends to bind to the filter structure during filtration.
In known devices, of the type referred to here, the contact between the absorbent and the membrane will limit the possible storage time of the device. The membrane structure is provided with wetting agents required to maintain the prameters for, the fluid transport through the membrane. During storage the absorbent will absorb these wetting agents and thus deteriorate the membrane.
The US: patent, U.S. Pat. No. 3,817,379 describes a disposable device for concentrating liquid specimens by filtration or ultrafiltration having a chamber with one wall formed of a membrane permeable to the liquid vehicle of the specimen and a layer of solid absorbent material pressed against the membrane outside of the chamber.
In this device the large effective filter area available at the start of the concentration is rapidly reduced as the concentration proceeds. The concentration speed will thus decrease. The use of a large initial filtration area also increases absorption losses on the membrane surface as mentioned above. This limits the volume range for the samples which can be used with this device and requires that alternative sizes of devices be offered.
If a larger volume than could initially be filled into the device is to be concentrated the device could of course be refilled. This would, however, mean that someone had to attend to the refilling which is not very convenient. Especially when several samples of liquid are concentrated simultaneously in parallel, possibly in a multiple concentrator device as shown in U.S. Pat. No. 3,817,379 having several compartments, refilling considerably increases the risk of cross contamination by mistake.
In a device of this type with one single membrane for several cells there is also the possibility of osmotic effect between adjacent: cells which would contaminate the retentates. Interaction between adjacent cells could of course also be due to poor sealing between the cells.
Due to the irregularities in the absorbent surface and the difficulty in obtaining a perfectly flat membrane surface, not all parts of the membrane are in contact with the absorbent material even if a resilient spongy material that compresses the absorbent sheet against the back of the membrane material should be used. The resultant reduction of absorptive surface area in contact with the membrane reduces the speed of filtration and the random contact points against the membrane surface further results in significant differences in concentration time between devices.
Another disadvantage with this device is that the relatively low hydrostatic pressure in the solute against the membrane surface will result in an extended period of time for the membrane and its support to be wetted out so that the capillary filtration can begin.
A further disadvantage is the capillary dimension of the filling and concentrate removal channel which requires the use of a specific glass Pasteur pipette which makes sample filling laborious and does not allow a precise quantitative recovery of final concentrate volume. The final concentrate must be first removed to a separate container before being again transferred for analysis by a conical quantitative pipette.
The problems of the prior art are overcome by the provision of a method and a device for carrying out said method as defined in the appended claims.
An advantageous embodiment of the invention will provide variability in effective membrane area so as to achieve the best relationship between concentration speed and protein absorption on the membrane surface.
The same embodiment will make it possible to separate the absorbent material from the membrane structure during transport and storage in order di to avoid drying out of the membrane wetting agents by the absorbent material.
It is an object of the invention to provide a concentrating device having a low volume filtration or concentration chamber preferably in the form of a flat, thin compartment, in the side wall of the membrane is mounted, arranged below a detachable sample reservoir which seals onto the concentration chamber by means of a conical or any other type of seal arrangement.
Whilst allowing increased flexibility for different sample volumes by changing the volume of the detachable reservoir without a change in membrane area this arrangement maximizes sample contact time with the total membrane surface during an extended period of the concentration. In the device according to the US patent above, the sample contact time with the total membrane surface is limit ed to the very first instant of the concentration procedure.
It is another object of the invention to provide improved hydrostatic pressure on the membrane surface to speed up the time required to wet out the membrane surface and supporting material and to assist the capillary action of the absorbent material during the initial stages of concentration.
It is another object of t invention to provide a filling aperture that supports the conical tip of a standard 10 ml pipette to provide easier filling in a single step.
It is another object of the invention to provide a concentrate recovery method that utilizes standard conical micro volume pipette tips to allow direct quantitative transfer of the concentrated sample without the need for an intermediate vessel.
It is another object of this invention to provide a concentrator which gives constant final volume in a separate concentrate pocket arranged below and efficiently separated from the active surface area of the membrane.
It is another object of the invention to maximize membrane yield to reduce manufacturing cost. The efficiency of the device will make it possible to use a much smaller membrane area for the same or shorter total concentration time as in a device according to the prior art.
It is another object of the invention to provide a multiple device in which the risk of cross contamination between adjacent cells is eliminated.
Further advantages and characteristics of the invention will be apparent from the following description.